The diarrheal disease cholera poses enormous health, economic, and political burdens worldwide. The causative agent of cholera, Vibrio cholerae, is endemic in aquatic habitats, but it is capable of infecting the human small intestine, causing a massive, life-threatening diarrheal response. To colonize the host and cause disease, V. cholerae must acquire essential nutrients, including iron, in the host environment. The predominant form of iron present in the small intestine is ferrous iron. Thus, it is critical to understand the mechanisms of ferrous iron uptake in V. cholerae. The major ferrous iron transporter in V. cholerae is Feo. The Feo system is widely distributed among all bacterial species, and has important functions in the virulence of several pathogenic species; nevertheless, very little is known about its structure and mechanism of transport. In V. cholerae, the Feo transporter is composed of three proteins, FeoA, FeoB, and FeoC. The membrane-embedded C-terminal domain of FeoB is likely to form the pore for iron transport, and, interestingly, its N-terminal domain has homology to small eukaryotic G proteins, suggesting a novel mechanism of transport. The roles of FeoA and FeoC are unknown. We recently demonstrated that the three Feo proteins associate to form a higher order complex in vivo. This represents the first structural analysis of the mature, membrane- embedded, active Feo complex in vivo. The goal of this proposal is to determine the overall structure of this large complex in order to build and test models for the mechanism of iron transport. In our first specific aim, we will determine the mass and stoichiometry of the native Feo complex. This will lay the groundwork for a structural model of the active Feo transporter. In aim 2, we will refine this model by delineating the sites of interaction within and between the members of the complex. We will then test the functional significance of these interactions for complex formation and iron transport activity. In aim 3, we will determine the source of energy for transport through Feo. These studies will significantly advance our knowledge of the structure and function of this important and unique iron transporter. Significantly, all our studies will be carried out using active, membrane-associated Feo complexes in vivo, giving our results an undeniable relevance over the in vitro studies that currently dominate the Feo field. As our previous work shows, V. cholerae is an ideal model organism for the study of Feo. We have already assembled most of the strains and reagents needed, and we, and our collaborators, have the expertise required to carry out the proposed experiments.